WO2010091051A2 - Utilisation thérapeutique de cellules progénitrices endothéliales spécialisées - Google Patents

Utilisation thérapeutique de cellules progénitrices endothéliales spécialisées Download PDF

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WO2010091051A2
WO2010091051A2 PCT/US2010/022996 US2010022996W WO2010091051A2 WO 2010091051 A2 WO2010091051 A2 WO 2010091051A2 US 2010022996 W US2010022996 W US 2010022996W WO 2010091051 A2 WO2010091051 A2 WO 2010091051A2
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ecfcs
cells
ischemic
ischemia
composition
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PCT/US2010/022996
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WO2010091051A3 (fr
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Robert I. Grove
Christine Smith-Steinhart
Paul A. Hyslop
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Endgenitor Technologies, Inc.
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Priority to JP2011548415A priority Critical patent/JP2012516853A/ja
Priority to US13/146,137 priority patent/US20120020925A1/en
Priority to EP10739045A priority patent/EP2393916A2/fr
Publication of WO2010091051A2 publication Critical patent/WO2010091051A2/fr
Publication of WO2010091051A3 publication Critical patent/WO2010091051A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/069—Vascular Endothelial cells
    • C12N5/0692—Stem cells; Progenitor cells; Precursor cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/44—Vessels; Vascular smooth muscle cells; Endothelial cells; Endothelial progenitor cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/04—Inotropic agents, i.e. stimulants of cardiac contraction; Drugs for heart failure
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K2035/124—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells the cells being hematopoietic, bone marrow derived or blood cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/13—Coculture with; Conditioned medium produced by connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
    • C12N2502/1305—Adipocytes

Definitions

  • the present invention relates generally to products and methods pertaining to the angiogenic and vasculogenic potential of highly proliferative endothelial progenitor cells, termed ECFCs, for various purposes including therapeutic use in mammals.
  • Ischemia is a medical condition characterized by insufficient oxygenation to any part of the body.
  • peripheral vascular disease is an ischemic condition caused by obstruction of arteries and blood flow to the leg.
  • PAD is particularly prevalent among the elderly, affecting approximately 10.3 million people in the U.S.
  • Many patients with PAD experience debilitating leg pain with exertion.
  • 25% of patients with PAD require partial amputation of one leg within a year of diagnosis. This represents over 100,000 people annually in the U.S. By five years, only half of these patients will still have both legs intact, and nearly 20% of the initial patients will have died from this or a related ischemic disease.
  • Ischemia can also lead to heart disease. About 1.2 million Americans suffer a heart attack every year, with approximately 40% resulting in death. Many heart attacks are traceable to coronary artery disease (CAD) which results in partial or complete obstruction of cardiac blood vessels. While roughly 60% of patients survive a heart attack, frequently there is permanent heart muscle damage. Even in the best-case scenarios, cardiologists are only able to save about 60% of cardiac muscle after a heart attack. According to NHANES the total prevalence of all forms of coronary heart diseases in the United States exceeded 13 million in 2003, at a staggering cost of $403 billion.( Heart Disease and Stroke Statistics -2008 Update: Circulation, 2008; 117 e25 - el46 ).
  • vasculogenesis occurs mainly during embryologic development, and is associated with endothelial colony forming cell (ECFC) migration and differentiation in response to local factors such as growth factors and extracellular matrix to form new blood vessels.
  • ECFC endothelial colony forming cell
  • Means for inducing vasculogenesis and angiogenesis may prove to be important in treating and/or preventing myocardial ischemia and other ischemic-related conditions. Areas of the myocardium following an ischemic episode become necrotic, the tissue dies and fibrotic scar tissue forms. Functional remodeling of fibrotic tissue may occur if a de novo blood supply can be established as a first step to providing nutrients and gas exchange capabilities to the necrotic tissue. Establishment of a de novo blood vessel network in an ischemic area that inosculates with existing surrounding vasculature of functional tissue is characteristic of the regenerative process. Once this process has occurred, local angiogenic processes provide an appropriate degree of vessel density as the tissue remodels and regains functionality.
  • Surgical interventions can be highly successful in treating various ischemias including myocardial ischemia.
  • coronary artery bypass surgery or angioplasty can, in most cases, reduce the symptoms of myocardial ischemia.
  • alternatives to surgical intervention are desirable for a variety of reasons including the potential to reduce medical costs, discomfort, and recovery time for patients.
  • pharmacological interventions are of increasing interest as a possible means to stimulate the body's ability to generate new blood vessels.
  • administering one or more growth factors such as FGF-I , FGF-2, FGF-5, PDGF-I , PDGF-2, VEGF, and IGF has received considerable research interest.
  • Other potential therapies include cell-based treatments which are of increasing interest for the treatment and/or prevention of ischemic disorders.
  • oxygen supply including but not limited to diseases and conditions of the myocardium, congenital heart deficit, heart valve disease, arrhythmia, left ventricular dilation, emboli, heart failure, coronary artery disease (CAD), angina, subendocardial fibrosis, left or right ventricular hypertrophy, myocarditits, myocardial infarction (MI), congestive heart failure (CHF), stroke, peripheral artery disease (PAD), wound healing and/or other ischemic diseases.
  • diseases and conditions of the myocardium congenital heart deficit, heart valve disease, arrhythmia, left ventricular dilation, emboli, heart failure, coronary artery disease (CAD), angina, subendocardial fibrosis, left or right ventricular hypertrophy, myocarditits, myocardial infarction (MI), congestive heart failure (CHF), stroke, peripheral artery disease (PAD), wound healing and/or other ischemic diseases.
  • CAD coronary artery disease
  • MI myocardial infarction
  • composition comprising highly proliferative endothelial colony forming cells comprising karyotypically normal
  • ECFCs for therapeutic purposes including treating an ischemic disorder in a patient in need of such treatment.
  • ECFCs including cryopreserved ECFCs
  • one or more other agents including cryopreserved or freshly isolated helper cells such as adipose stromal cells (ASCs), pharmaceutical carriers, excipients, buffers, proteins, peptides, matrix materials, and/or other agents for use in treating ischemic disorders.
  • ASCs adipose stromal cells
  • Still another object of the invention relates to methods and/or processes for producing, improving and/or enhancing the angiogenic potential of ECFCs.
  • the invention relates to a composition of matter comprising karyotypically normal ECFCs.
  • the present invention relates to a product-by-process comprising karyotypically normal ECFCs.
  • the present invention relates to a method for treating a patient with an ischemic disease including myocardial infarction (MI), coronary artery disease (CAD), congestive heart failure (CHF), stroke, peripheral artery disease (PAD), and other ischemic disease including myocardial ischemia, chronic myocardial ischemia and acute myocardial ischemia comprising administering an effective amount of ECFCs to stimulate angiogenesis and/or vasculogenesis.
  • MI myocardial infarction
  • CAD coronary artery disease
  • CHF congestive heart failure
  • PED peripheral artery disease
  • the present invention relates to a method for increasing blood flow or perfusion to a site in a patient in need thereof, including a site of ischemic injury, comprising administering ECFCs.
  • the present invention relates to a method to reverse, limit or prevent ischemic damage and/or tissue death in a patient in need thereof by inducing angiogenesis and/or vasculogenesis comprising administering an effective amount of
  • the present invention relates to a method to reverse, limit or prevent vascular damage associated with an ischemic disease or condition comprising administering an effective amount of ECFCs.
  • the present invention relates to a method to reverse, limit or prevent cardiac cell apoptosis comprising administering ECFCs.
  • the present invention relates to a method to reverse, limit or prevent the effects of ischemic disease comprising administering ECFCs in combination with one or more helper cell types, or other source of smooth muscle cells and/or pericytes, preferably also including a suitable matrix material to a patient in need thereof.
  • the present invention relates to a method for inhibiting or reducing fibrosis associated with ischemia including but not limited to myocardial ischemia by administering ECFCs alone or in combination with one or more suitable helper cell types and a matrix material to a patient in need thereof.
  • the invention relates to administering ECFCs alone or in combination with helper cells including but not limited to those selected from the group consisting of adipose stromal cells, bone marrow mononuclear cells, and endometrial mesenchymal cells, or Wharton's jelly mesenchymal cells to enhance blood flow and reverse, limit or prevent ischemic diseases including myocardial infarction (MI), congestive heart failure (CHF), stroke, peripheral artery disease (PAD), and other ischemic disease.
  • helper cells including but not limited to those selected from the group consisting of adipose stromal cells, bone marrow mononuclear cells, and endometrial mesenchymal cells, or Wharton's jelly mesenchymal cells to enhance blood flow and reverse, limit or prevent ischemic diseases including myocardial infarction (MI), congestive heart failure (CHF), stroke, peripheral artery disease (PAD), and other ischemic disease.
  • MI myocardial in
  • the present invention relates to administration of ECFCs in combination with one or more other pharmacological agents including but not limited to, ⁇ -blockers, diuretics, Ca-channel blockers and ACE inhibitors to treat or prevent a disease including myocardial infarction (MI), congestive heart failure (CHF), stroke, peripheral artery disease ( PAD), and ischemic disease or condition.
  • MI myocardial infarction
  • CHF congestive heart failure
  • PAD peripheral artery disease
  • the present invention relates to administering to a patient in need thereof a composition comprising ECFCs alone or in combination with helper cells and optionally containing a matrix material in conjunction with an adjunct procedure including but not limited to prosthetic device(s) including stents and vascular prosmeses.
  • the present invention relates to a method for treating myocardial infarction (MI), congestive heart failure (CHF), stroke, peripheral artery disease (PAD), coronary artery disease (CAD), and ischemia comprising administering
  • MI myocardial infarction
  • CHF congestive heart failure
  • PDA peripheral artery disease
  • CAD coronary artery disease
  • ischemia comprising administering
  • ECFCs in combination with one or more other biologically active agents including proteins, peptides, and growth factors.
  • the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a therapeutically effective amount of ECFCs alone or in combination with one or more other agents including helper cells and/or other pharmacological agents including but not limited to growth factors, ⁇ -blockers, diuretics,
  • Ca-channel blockers Ca-channel blockers, ACE inhibitors, and optionally also including a suitable matrix material.
  • Figure IA top panel.
  • ECFC -treated animals show a significant improvement in reduction in diameter compared with negative control, closely approximating normal diameter.
  • Figure 1 B bottom panel.
  • ECFC-treated animals show an approximate 50% improvement over control animals.
  • Figure 2 Capillary density in stained sections of infarcted rat heart at two and four weeks after induction in control (left panels, top and bottom) and ECFC-treated animals (right panels, top and bottom). [035
  • FIG. Histological section of experimentally induced ischemic rat myocardium injected with mixture of ECFCs, ASCs and Extracel m matrix material.
  • Arrows labeled * 'B" identify human nuclei in cells that have integrated into rat blood vessel linings.
  • Figure 7 Histological section of experimentally induced ischemic rat myocardium taken from a portion of heart adjacent to site of injection.
  • HPP-EPC' refers to a subclass of endothelial progenitor cells isolated from cord blood having high proliferative potential as decribed in WO/2005/078073.
  • ECFCs refers to endothelial colony forming cells isolated from placenta cord, cord blood, or other suitable source.
  • ECFCs exhibit a normal karyotype.
  • Karyotypically normal ECFCs may be obtained by expansion of HPP-EPC according to methods described herein from clones having a normal karyotype.
  • a commercially available source of preferred ECFCs are
  • anastomose refers to a biological process, or result thereof, in which a physical connection is made between tubular structures, including blood vessels.
  • the term "therapeutically-effective" means providing a clinically relevant benefit to a patient, e.g. in treating myocardial ischemia as measured, for example, by increased cardiac perfusion, reduced angina, and/or neovascularization.
  • ASC refers to adipose tissue stromal cells including cells from a human or other mammalian sources.
  • angiogenesis means any physiological process involving the growth of new blood vessels from pre-existing vessels [046J
  • vasculogenesis as used herein is the term used for spontaneous formation of new blood vessels from vascular cells
  • arteriogenesis refers to an increase in the diameter of existing arterial vessels.
  • normal karyotype or “substantially normal karyotype” or “karyotypically normal” as applied herein to a cell or population of cells means that the proper number of chromosomes are present and not noticeably altered. In a population of cells, greater than about 95%; more preferably greater than about 98%; still more preferably greater than about 99% of cells exhibit this characteristic as detected by any suitable means.
  • karyotypically normal ECFCs i.e. ECFCs®
  • matrix or “matrix material” or “pharmaceutically acceptable matrix” or “extracellular matrix” means any suitable biocompatible polymer including synthetic or recombinantly engineered peptide or protein polymers.
  • polymer matrices include but are not limited to matrix materials containing collagen and/or fibronectin, hydrogels containing a combination of native or modified hyaluronan, heparin, and gelatin, crosslinked by polyethylene glycol diacrylate, PEGDA.
  • a matrix material is admixed with cells prior to administration which is thought to concentrate cells at a particular location.
  • co-administer refers to therapeutic administration to a subject including a human patient of more than a single agent either simultaneously or sequentially.
  • co-administration of two different cell types could involve a single administration of a dosage form having two different cell types in admixture, or one or more administrations to a patient of a dosage form having one cell type which precedes administration of a dosage having the other cell type.
  • helper, or “helper cells” or “helper cell population” is used herein to mean any cell population, cell-based material, or composition of matter that when combined with ECFCs facilitates angiogenesis and/or vasc ⁇ logenesis when administered to a mammal in need thereof.
  • helper or “helper cells” are believed to provide a suitable source of pericytes and/or smooth muscle cells that enhance angiogenesis and/or vasculogenesis when administered or co-administered in vivo to a mammal including a human.
  • a suitable helper includes but is not limited to adipose stromal cells and/or endometrial mesenchymal cells alone or in combination with one or more other helper cell type(s).
  • helper cells is intended to broadly include any suitable source of smooth muscle cells and/or pericytes.
  • ischemic disease refers to diseases and/or condition characterized by reduced oxygenation to any tissue in the body such as, but not limited to, ischemic heart disease, transient ischemic attack (TlA), cardiac ischemia, stroke, reperfusion injury, bowel ischemia, intestinal ischemia, peripheral artery disease, critical limb ischemia, mesenteric ischemia, brain ischemia, leg ischemia, myocardial infarction, peripheral vascular disease, coronary artery disease, angina, wound healing, renal artery disease, diabetic ulcer healing, congestive heart failure, and hepatic ischemia.
  • TlA transient ischemic attack
  • cardiac ischemia stroke
  • reperfusion injury bowel ischemia
  • intestinal ischemia intestinal ischemia
  • peripheral artery disease critical limb ischemia
  • mesenteric ischemia mesenteric ischemia
  • brain ischemia leg ischemia
  • myocardial infarction peripheral vascular disease
  • coronary artery disease angina
  • Ischemia can be caused by a number of conditions including but not limited to anemia, stroke and atherosclerosis.
  • Multiple diseases result from ischemia including, for example, cerebrovascular ischemia, renal ischemia, pulmonary ischemia, limb ischemia, myocardial ischemia, and ischemic cardiomyopathy.
  • cerebrovascular ischemia renal ischemia
  • pulmonary ischemia pulmonary ischemia
  • limb ischemia myocardial ischemia
  • myocardial ischemia myocardial ischemia
  • ischemic cardiomyopathy ischemic cardiomyopathy
  • the terms "dosage”, “amount”, or “number,” are used essentially interchangeably to indicate the quantity of ECFCs, preferably ECFCs®, or other cells to be administered to achieve clinical benefit.
  • the methods and pharmaceutical compositions of the invention can be used to promote arterial vascular growth, for example in the treatment or prevention of conditions associated with ischemia. Such conditions include, but are not limited to, stroke or heart attack.
  • the products, methods, and compositions of the invention can be used to accelerate wound healing, promote vascularization of surgically transplanted tissue, and enhance the healing of a surgically-created anastomosis.
  • 055] The methods and compositions of the invention are effective for enhancing blood flow to biological tissues, including treating a patient suffering from, or at risk of suffering from, ischemic damage to an organ or tissue including but not limited to myocardial tissue.
  • Reduced blood supply to a tissue can be caused by a vascular occlusion, resulting from, for example, arteriosclerosis, trauma, or surgical procedure. Determining whether a tissue is at risk of or has already been affected by ischemic damage due to vascular occlusion is readily ascertainable using any suitable technique known to the skilled artisan including a variety of imaging techniques (e.g., radiotracer methodologies, such as 99 mTc-sestaraibi, x-ray, and MRI angiography) and physiological tests.
  • imaging techniques e.g., radiotracer methodologies, such as 99 mTc-sestaraibi, x-ray, and MRI angiography
  • Induction of vascular growth in a tissue affected by or at risk of being affected by ischemia is expected to prevent, treat or reverse ischemia, regardless of its origin, in organs or tissues including, but not limited to, brain, heart, pancreas, or limbs.
  • HPP-EPC represent a highly proliferative s ⁇ bpopulation of endothelial progenitor cells (EPC) distinct from endothelial cell colony forming units (CFU-ECs) and from endothelial outgrowth cells (EOCs).
  • EPC endothelial progenitor cells
  • CFU-ECs endothelial cell colony forming units
  • EOCs endothelial outgrowth cells
  • HPP-EPC express cell surface antigens that are characteristic of endothelial cells including CD31 , CD 105, CD 146, and CD 144, but do not express antigens that are characteristic of hematopoietic cells, such as CD45 and CD14.
  • HPP-EPC are obtained by passaging cells at 90% to 100% confluence as described in WO 2005/078073. A. Isolation of Karyotypically Norma! ECFCs
  • the present invention relates to a population of clonally purified, high proliferative endothelial colony forming cells which have a substantially normal karyotype.
  • ECFCs having a substantially normal karyotype can be produced from various tissue sources including cord blood by any suitable method including methods described herein.
  • Methods pertaining to this aspect of the invention reduce the incidence of abnormal karyotype in expanding endothelial progenitor cells during passage. It is believed that enhanced karyotypic normalcy in ECFC cells may provide a safer and/or more effective clinical outcome.
  • a method for producing ECFCs involves passaging endothelial progenitor clones before they reach confluence. For example, cells are passaged when they reach less than about 90% confluence, or between about 70% to less than 90% confluence; or less than about 80% confluence; more preferably from about 75% to about 80% confluence prior to trypsinization and passage.
  • Another aspect of the invention relates to inducing blood flow to a tissue in need thereof including ischemic tissue such as, but not limited to, ischemic myocardium comprising administering a mixture of ECFCs and helper cells, e.g. ASCs.
  • ECFCs are admixed with one or more different helper cell types and injected into NOD/SCID mice.
  • ECFCs® are mixed with one or more helper cell types and MatrigelTM (GFR-Matrigel:HC-Matrigel) in a 3:1 ratio of ECFCs® to helper cells and a 50:50 admixture of cells to matrix material prior to injection subcutaneously into NOD/SCID mice.
  • Cells and matrix admixtures were harvested 14 days after injection and processed for histological examination. Human blood vessel formation was examined by H&E staining and by imm ⁇ nochemistry using anti-human CD31/anti-h ⁇ man nuclear matrix staining.
  • NON/SCID mouse model when ECFCs® are administered with ASCs, alone or in combination with other helper cell types. New blood vessel formation was also detected at low levels when ECFCs® were admixed with HASMC and CD 133+ (unexpanded) (Table 4).
  • One embodiment of the present invention relates to administering an effective amount or dosage of ECFCs, preferably karyotypically normal ECFCs (e.g. ECFCs®), to restore and/or improve blood flow to ischemic tissues including myocardial tissues.
  • ECFCs are mixed wkh helper cells, and a suitable matrix. No evidence could be found that ASCs, mesenchymal cells of any source, or ECFCs when administered alone, or ECFCs in combination with a matrix material, induce robust vasculogenesis in comparison to ECFCs in the presence of one or more appropriate "helper" cells.
  • Helper cells contribute to and synergize with ECFCs in promoting vasculogenesis and are believed to provide a source of smooth muscle cells and/or pericytes, which are believed to stabilize nascent tube formation.
  • Suitable helper cells include but are not limited to an appropriate source of smooth muscle cells and/or pericytes, for example, ASCs and mesenchymal endometrial cells (Cryo-Cell, Inc.).
  • the effect of enhancing vasculogenesis by administering ECFCs in combination with helper cells is facilitated by administering the cells in a suitable matrix material. It is believed that the matrix helps concentrate the cells and prevent or reduce cell migration or dilution at a site of administration (e.g. injection).
  • Rut Myocardial Ischemia Model [071 j ECFCs ⁇ are tested in a rat myocardial ischemia model to assess their ability to restore blood perfusion and improve heart function in ischemic tissue (Studies 1 - 3).
  • Test animals receive ECFCs® alone, ASCs alone, ECFCs® + ASCs, or saline.
  • ECFCs® are injected in a liquid carrier into the perimeter of the ischemic area in four aliquots of 20-50ul each. Each animal receives a total of approximately 10* cells/kg bodyweight
  • ECFCs* and ASCs are mixed together with a suitable matrix material (Matrigel ⁇ /Hydrogel) prior to administration to assess possible synergy in the restoration of blood flow and heart function. Endpoints are measured at 14 days (Studies 1-3) and 28 days (Study 2) post-administration by echocardiography measurement in live animals. After sacrifice, blood vessel size and density in the ischemic tissue is evaluated by histological examination. In addition, histological samples are stained to reveal human cells in the endothelial lining of cardiac blood vessels.
  • EGMTM-2 complete endothelial cell culture media containing various growth factors including VEGF.
  • ADSC media complete adipose-derived stem cell media containing 10% fetal calf serum
  • DMEM fetal calf serum
  • bovine serum albumin 0.5% bovine serum albumin and 10% dimethylsulfoxide.
  • ECFCs® and ASCs Freeze media Fetal calf serum containing 5% dimethylsulfoxide.
  • Cord blood mononuclear cells are prepared from donated umbilical con- blood by standard methods, and seeded at 5 x 10 7 cells/well in collagen-coated 6-well plates containing 4mL complete EGM*-2 media (Lonza) and placed in a humidified 37°C; 5% (v/v) COa incubator. Every 24 h for 7 days, and then every 2 days, the media is gently replaced On day 12, clones are plucked from the wells and the colony dissociated with trypsin/EDTA. Each colony is seeded into a single well containing 2mL complete EGM*-2 and returned to the incubator.
  • MNCs Cord blood mononuclear cells
  • Cells are expanded into flasks when the cells are less than about 75-80% confluent, usually between 4 to 7 days.
  • the clonal selection and re-plating process selects the HPP ECFCs® subpopulations of adherent cells from the MNCs in cord blood.
  • expanded ECFCs® are cryopreserved in aliquots of 10 6 cells per vial ("Passage 3" vials). Passage 3 vials are thawed and expanded to passage 4, yielding approximately 10 8 cells, and then cryopreserved in appropriate aliquots (ECFCS*). Passaging, Expansion and Cryopreservation of ASCs
  • Cryopreserved ASCs are removed from liquid nitrogen storage and warmed in a 37°C water bath until crystals are no longer present in the vials.
  • the ASCs are transferred from the cryovial to 2OmL ASC complete media and plated onto a collagen coated T 150. Cells are incubated overnight at 37°C in 5% CO 2 humidified tissue culture incubator. To remove residual DMSO, tissue culture media is removed from the cells 18 brs later, and 20 mL of fresh ASC complete media is added to the cells. ASCs are cultured until they are 85% confluent at which time the cells are passaged. Adherent ASCs are replenished with fresh ASC complete media every other day.
  • the ASCs are cryopreserved in aliquots of 10 6 cells per vial.
  • the cells are slowly cooled to -70°C using a controlled rate freezer and stored in liquid nitrogen.
  • Preparation of Cells for Injection Preparation of ECFCs® in liquid medium (Studies 1 & 2)
  • ECFCs® are removed from liquid nitrogen storage, and quickly warmed in a 37°C water bath. Cells are transferred to pre-warmed, complete EGMTM-2 and pelleted at 300-400xg for 7-10 minutes. The supernatant is removed and cells are resuspended in complete media to be cultured in suspension for 1-2 hours in a humidified 37°C; 5% (v/v) CO 2 incubator. Cells are centrifuged at 300-400xg for 7-10 minutes, the supernatant removed, and the cells are resuspended in Dulbecco's PBS at concentrations to give a dose of 10 6 cells/kg. The resuspended cells are drawn up in a 3OG Hamilton syringe for injection. Preparation of EXTRACEL*/ASC/ECFCs* Admixture (Study 3)
  • Gelin-Sd> and HeprasikD 0.5ml of DQ water is added to the vial of ExtraLink®. The vials are incubated for an additional 30 minutes at 37°C. ImI of Gelin-S® is combined with ImI of Heprasil® in a 15ml conical flask. 255 ⁇ l of Extracel-HP® + cells are prepared per injection site.
  • Extracel-HP® mixture 165 ⁇ l of appropriate Extracel-HP® mixture is added to 40 ⁇ l of cells in EGM-2 and mixed well. 50 ⁇ l of ExtraLmk® is added to each sample to initiate solidification.
  • Extracel-HP® begins to solidify, and the matrix infused with cells is drawn up in a syringe for injection.
  • Test articles are administered by intramyocardial injection via a 100 ⁇ l Hamilton syringe and 28 gauge needle.
  • the test and vehicle/control articles are administered once on Day 0, as 4 separate injections into the left ventricular free wall of the heart using a range of cell dosages and volumes. For example, approximately 1-2 x 10 6 cells are administered per 100 ul volume.
  • J095J A midline sternotomy is performed on anesthesized animals.
  • the pericardium is opened and the test article or vehicle is injected at four separate sites into the left ventricular anterior free wall of the heart.
  • the LAD is identified and then is either permanently occluded with a suture, or is occluded termporarily for about 1 hour.
  • Occlusion is verified by blanching of the myocardium and confirmation of characteristic changes of ischemia on the ECG waveform (e.g. ST elevation).
  • the suture is removed and the infarcted area is allowed to reperfuse. Following reperfusion the test article is injected as described above and the thorax closed.
  • Sections are stained with a collagen stain (e.g., picrosirius red) to assess collagen density as a measure of infarct area. Histomorphometry is performed and the following measurements taken: I) the inner LV circumference, 2) the outer LV circumference, 3) the outer and inner infarct arc, and 4) the area of the infarct. Data are recorded as a percent of area of LV infarcted. Histochemistry Assessment of capillary density:
  • a collagen stain e.g., picrosirius red
  • Sections are stained with anti-rat vWF (von Willebrand's Factor). Five microscopic fields (40Ox magnification) from each section, cut perpendicular to the long axis of the cardiac muscle fibers, are acquired and digitized. The total number of capillaries in each field is counted using Image-Pro Plus software (Media Cybernetics, Rockville, MD) and capillary density (capillaries/mm 2 ) calculated. Assessment of human blood vessel formation:
  • Echocardiographic evaluation of heart function is performed immediately before surgery and two weeks later, prior to sacrifice.
  • a 2-dimensional short-axis view of the left ventricle (LV) is obtained at mid-papillary and apical levels.
  • M -mode tracings are recorded through the anterior and posterior LV walls to allow delineation of wall thickness and motion in infarcted and non-infarcted regions of the heart. The results are recorded and LV mass determined.
  • Relative anterior wall thickness, posterior wall thickness, and LV internal dimensions are measured, preferably from at least three consecutive cardiac cycles. Endocardial fractional shortening and midwall fractional shortening are used as indices to estimate LV systolic function.
  • Echocardiography measurements including left ventricular pressure and contractility in cell-treated animals are not significantly different from negative controls.
  • LVEDD left ventricular end diastolic diameter
  • anterior wall thickness As another test of the effectiveness of the treatment, measurements are also taken of anterior wall thickness. Significant reduction in anterior wall thickness occurs in rats that undergo coronary artery ligation followed by saline injections. However, rats treated with ECFCs® show significant increase in anterior wall thickness. In contrast, no differences are observed in the posterior wall thickness in any of the treated groups compared to controls (data not shown).
  • Ejection fraction is an additional independent measure of heart function, routinely estimated in short-axis mode from digital images at the mid-papillary level of the heart.
  • ejection fraction is determined at the apical portion of the heart. Animals treated with ECFCs® show an improvement in ejection fraction
  • ECFCs have been shown to undergo vasculogenesis in a subcutaneous collagen/fibronectin implant in the NOD/Scid immunodeficient mouse, with concomitant inosculation as evidenced by blood vessels in the implant that contain and circulate host blood. Endothelial cells have been shown to form vascular networks in vivo (Yoder et al.. Blood, 109, 1801-1809, 2007) and in healthy tissue (Malero-Martin et al Circulation Res. (2008); 103 194-202).
  • ECFCs are karyotypically normal (e.g. ECFCs ⁇ ).
  • ECFCs® and ASCs human adipose tissue stromal cells
  • an extracellular matrix for example Matrigel® or Extracel® prior to administration, for example, by injection into an ischemic tissue including an ischemic region of myocardium.
  • Injections of an ECFC ⁇ /ASCVmatrix composition into ischemic rat myocardium results in the formation of stable human blood vessels within the ischemic area of the rat heart demonstrating that vasculogenesis occurs within the ischemic myocardial environment.
  • a control histological section of healthy human colon, showing CD31 staining (human anti-CD31 bound to a red dye) and nuclei staining (human anti-nuclear matrix protein bound to a brown dye) is shown in Figure 4.
  • a histological section of normal rat myocardium injected with a mixture of ECFCs®, ASCs, and Extracel® showing human anti ⁇ CD31 and human nuclei in vessels is shown in Figure 5.
  • the vessels contain rat red blood cells, demonstrating that the human vessels have inosculated with the rat blood supply.
  • Figure 6 shows the presence of human blood vessels in the ischemic rat myocardium injected with a mixture of ECFCs ⁇ , ASCs, and Extracel ⁇ , thereby establishing that human endothelial cells line the blood vessels.
  • the intimal areas of the blood vessels are identified by unlabeled arrows in Figure 6, showing the line that divides the lumen of the blood vessel from the surrounding tissues.
  • Arrows labeled "B" in Figure 6 identify human nuclei in cells that are integrated into the intimal lining of the blood vessel ⁇ - that is endothelial cells of human origin and therefore derived from ECFCs®. Other human nuclei are present which are not associated with blood vessels.
  • one aspect of the invention relates to promoting an angiogenic effect by administering ECFCs alone, preferably in a liquid carrier.
  • the invention relates to inducing a vasculogenic effect, by administering or co-administering ECFCs in admixture with one or more helper cell types, preferably in a suitable matrix material.
  • the ECFCs have a normal karyotype; most preferably ECFCs are ECFCs®.
  • This aspect of the invention is expected to provide a variety of therapeutic options to maximize clinical benefit according to the need of each patient.
  • Different physiological effects can be induced by administering ECFCs alone or in combination with helper cells. Treatments can be targeted at increasing angiogenesis alone or in promoting both angiogenesis and vasculogenesis.
  • a paracrine angiogenic effect can be induced by administering ECFCs* alone directly into injured tissue surrounding an infarct zone.
  • angiogenic and vasculogenic effects can be induced by co-administering ECFCs* and one or more helper cell types, e.g. ASCs in a relevant non-toxic matrix. Inducing vasculogenesis and/or angiogenesis is expected to lead to more effective treatments through restoration of blood flow, accelerated tissue remodeling, and recovery of function in ischemic tissues.
  • the methods of the present invention provide improved therapeutic methods for treating diseases associated with reduced blood flow and/or insufficient perfusion, for example, myocardial infarction (Ml), congestive heart failure (CHF), stroke, peripheral artery disease (PAD), and ischemic disorders including myocardial ischemia generally and more specifically acute myocardial ischemia, chronic myocardial ischemia, myocardial infarction, CAD, left ventricular dysfunction, and end-stage ischemic heart disease.
  • Suitable patients include but are not limited to those with severe ischemic heart failure and chronic coronary artery disease.
  • the present invention relates in part to the use of ECFCs, alone or in combination with helper cells and/or other agents such as a suitable matrix material and growth factors, for therapeutic purposes.
  • ECFCs are karyotypically normal (e.g. ECFCs ⁇ ) that are administered as an admixture with helper cells, preferably ASCs and a matrix material. It is anticipated that use of karyotypically normal ECFCs in clinical applications will result in enhanced efficacy and/or safety.
  • ECFCs alone or in combination with one or more helper cell types are administered to a patient in need thereof to prevent or treat diseases that include, for example, myocardial infarction, congestive heart failure, stroke, peripheral artery disease, and ischemic disease, including limb ischemia or myocardial ischemia including acute and chronic myocardial ischemia.
  • diseases that include, for example, myocardial infarction, congestive heart failure, stroke, peripheral artery disease, and ischemic disease, including limb ischemia or myocardial ischemia including acute and chronic myocardial ischemia.
  • ECFCs® are administered as an admixture with ASCs and matrix material to a site at or near an ischemic region of tissue.
  • ECFCs may be admixed with adipose stromal cells and a matrix material, e.g.
  • Extracel® for purposes of administration to an ischemic region of the heart.
  • the composition and method may also further include one or more growth factors including but not limited to VEGF, bFGF, PDGF-I, PDGF-2, ⁇ GF, FGF- 1 , FGF-2.
  • ECFCs are combined with ASCs and the combination mixed with a suitable matrix material, e.g. Extracel-HP®, Gelfoam® (Pharmacia & Upjohn), or a collagen/fibronectin mixture.
  • ECFCs and ASCs may be combined in a ratio of 10:1 to 0.5: 1 , respectively; alternatively in a ratio of 5: 1 to 1:1; preferably in a ratio of 3: 1 to 2: 1.
  • a cell-based composition comprising ECFCs; or ECFCs and helper cells; or ECFCs, helper cells, and a matrix material are used in conjunction with other treatment options known to the skilled artisan including the use of stents or other vascular prosthetic devices used in treating ischemia disorders including myocardial ischemia.
  • ECFC compositions contemplated in this aspect of the invention may be administered before, during, or after a procedure that places such a device(s). (0122]
  • the methods of the invention are expected to induce blood vessel formation and improve blood flow thereby providing clinical benefit to patients in need thereof.
  • the present invention is expected to reduce the risk of and/or prevent myocardial infarction and/or reduce post-infarction damage to heart muscle, for example scarring and fibrosis.
  • the methods are also expected to reduce the risk and/or damage from ischemia, stroke, congestive heart failure and peripheral artery disease by inducing vessel formation and increasing blood flow to a tissue(s) and/or organ(s) in need thereof.
  • a patient in need receives an allogeneic transfer of ECFCs, alone or in combination with one or more other agents including helper cells, and/or growth factors and/or matrix materials, either systemically or by direct injection, into an ischemic area of tissue, for example, an ischemic area of the heart.
  • ECFCs are karyotypically normal; in the most preferred embodiment ECFCs are ECFCs®.
  • [0124J ECFCs can be delivered into the ischemic myocardium either by invasive or noninvasive means.
  • ECFCs can be administered by systemic infusion or by local transplantation at an ischemic site in the myocardium or region surrounding an ischemic site.
  • ECFCs are administered by injection transendocardially or trans-epicardially, allowing the cells to penetrate the protective surrounding membrane.
  • a preferred embodiment of the invention includes use of a catheter-based delivery of ECFCs for transendocardial injection.
  • the use of a catheter provides a less invasive method of delivery, avoiding the need to open the chest cavity and provides for quicker recovery.
  • cells are injected through a cardiac catheter into the wall of regions of the heart that are ischemic. Cells may also be injected into healthy surrounding tissue regions.
  • treatment comprises one or more injections of cells to a plurality of sites at or near a site of ischemic injury.
  • Invasive means include, but are not limited to, epicardial injection of cells into a surgically exposed heart directly into an ischemic area, an infarcted area, or into viable myocardial tissue surrounding diseased areas.
  • ECFCs to damaged myocardium
  • catheter-based rransendocardial injection which provides the benefit of less invasiveness and the ability to visually map the heart and determine the best place to inject cells.
  • hibernating myocardium may be a preferred target for this type of procedure.
  • An appropriate dosage of ECFCs to administer or deliver to a patient according to the present invention will depend on the particular patient, the condition being treated and will involve such factors as mode of administration, patient bodyweight, and severity of the disease or condition being treated.
  • an effective dosage would fall within a range of about 1 x I O 5 to about 1 x 10 7 ECFCs per kg bodyweight; or about I x IO 6 to about 1 x IO 7 cells per injection site.
  • the ratio of ECFCs to helper cells is about 1 : 1 to about 20: 1 ; preferably about 2: 1 to about 3:1.
  • the ratio could also be about 6:4 to about 1.9.
  • a therapeutically effective dose of ECFCs cells is applied, delivered, or administered to the heart or implanted into the heart of a patient in need thereof.
  • An effective dosage is an amount or number sufficient to achieve a beneficial or desired clinical result.
  • An effective dose can be administered in one or more administrations. However, the precise determination of an effective dose will be based on factors individual to each patient, including bodyweight, age, size of the infarcted area, and amount of time elapsed since occurrence of the damage. The treating physician, surgeon, or cardiologist, would be able to determine the number of cells which would constitute an effective dose without being subject to undue experimentation, from this disclosure and the knowledge in the art. (0127]
  • ECFCs are delivered to the heart, specifically to the border area of the infarct. As one skilled in the art would be aware, the infarcted area is generally visible to the naked eye, allowing targeted placement of stem cells to the infarcted area.
  • the present invention also contemplates methods and kits in which ECFCs, preferably karyotypically normal ECFCs such as ECFCs®, are combined with or coadministered with one or more other agents including, for example, CD34 * cells, ⁇ - blockers, diuretics, Ca-channel blockers, ACE inhibitors, proteins or peptides and growth factors.
  • co-administration involves administering ECFCs, sequentially, concurrently, or simultaneously with one or more other agents.
  • kits which includes a container with ECFCs, optionally also including one or more containers with other agents selected from the group consisting of helper cells, matrix material, CD34' cells, ⁇ -blockers, diuretics, Ca-channel blockers, ACE inhibitors, proteins or peptides, and growth factors.
  • a kit according to the present invention may also include devices such as a stent(s), catheter, or syringe.
  • Another embodiment relates to the ability to directly covalently attach proteins , peptides growth factors, cytokines and antibiotics via a reactive thiol contained within a modified version of Extracel® , Heprasil® a combination of thioi-modified hyaluronan, HA, and thiol- modified heparin), Gelin-S®(thiol-modified gelatin), and Extralink® (a thiol-reactive crosslinker, polyethylene glycol diacrylate, PEGDA).
  • Extracel® Heprasil® a combination of thioi-modified hyaluronan, HA, and thiol- modified heparin
  • Gelin-S® thiol-modified gelatin
  • Extralink® a thiol-reactive crosslinker, polyethylene glycol diacrylate, PEGDA
  • MNCs mononuclear cell fraction
  • P3 clones When P3 clones are ready to be passaged they are frozen and quality control testing (QC) is performed on each clone including Matrigel® tube formation, cell surface markers, and expansion. Clones that pass the QC tests are expanded to P4 via Tl 50 flasks or roller bottles and thereafter frozen in liquid nitrogen. Each batch of frozen P4 cells is tested for mycoplasma and bacterial contamination and is karyotyped. ECFCs are cryopreserved in vapor phase liquid nitrogen.
  • QC quality control testing
  • a 60 year old male patient presents with chest pain induced by moderate exercise (angina) having a blood pressure of 360/90, a heart rate of 90 and a bodyweight of 210 Ib.
  • Clinical examination including ETT (exercise treadmill testing) and AECG (ambulatory electrocardiogram) as well as serum lipid profiling (showing elevated LDL) leads to a diagnosis of CAD with myocardial ischemia.
  • the patient is placed on 81 mg daily aspirin, a beta-blocker, a fat-restrictive diet and an aggressive treatment with a statin drug (Lipitor 80 mg/d) and 2 gm fish oil daily to lower his elevated triglycerides and LDL cholesterol levels. After 6 months treatment the patient still experiences angina wilh moderate level activity.
  • the patient is scheduled for intracardial administration of an ECFC-based composition
  • an ECFC-based composition comprising a mixture of ECFCs®, ASCs and a suitable matrix material in a 2:1 ratio of ECFCs® : ASCs at a concentration of about 1 x 10 7 cells/ml.
  • the cells are mixed with the matrix immediately prior to injection.
  • Cells are administered to the patient using a cardiac catheter.
  • About 100 ⁇ l of the cell-matrix mixture is injected at each of four roughly equivalently distributed places in the ischemic region, providing about 1 x 10 6 cells per site of injection.
  • Post-operative MSCT scans at 2 and 6 months show significantly improved cardiac blood flow and the patient reports that exercise no longer induces angina.

Abstract

L'invention concerne des produits, des procédés et des procédés thérapeutiques pour restaurer le flux sanguin vers des tissus présentant un risque d'ischémie ou déjà ischémiques, par induction d'une angiogenèse et/ou d'une vasculogenèse dans des tissus le nécessitant, par administration de cellules formant des colonies endothéliales seules ou en combinaison avec d'autres types de cellules et/ou agents. Dans un aspect, l'invention est utile pour induire une angiogenèse et/ou une vasculogenèse chez des patients souffrant d'une maladie ischémique, en particulier, une maladie cardiaque ischémique, et d'autres affections vasculaires ischémiques.
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KR20220143379A (ko) * 2021-04-16 2022-10-25 경희대학교 산학협력단 중간엽 줄기세포 및 혈관내피전구세포를 유효성분으로 포함하는 폐쇄성 혈관질환 또는 이의 합병증 예방 또는 치료용 조성물

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EP2776057A4 (fr) * 2011-11-07 2015-09-23 Hina W Chaudhry Procédés de réparation cardiaque
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EP3755354A4 (fr) * 2018-02-21 2022-01-19 Indiana University Research and Technology Corporation Compositions et procédés pour le traitement ou la prophylaxie d'un trouble de perfusion

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